Peripheral induction of antigen-specific Foxp3+CD4 T cells, or regulatory T cells (Tregs), is a potential pathway to treat autoimmune diseases. It is also represents an undesirable outcome of the immune response, which limits recovery from chronic infections and malignancies. Early work has shown that Treg induction is favored by suboptimal or low-dose antigen stimulation. More recently, others and we have demonstrated that mTOR blockade during antigen activation favors peripheral Treg induction. Similarly, other means of cellular metabolic stress during antigen activation, e.g., L-tryptophan deprivation, also lead to induction of anergic and Treg phenotypes. One common feature of these examples is inhibition of cap-dependent mRNA translation. Of course, it is well recognized that a small fraction of genes can actually increase their expression in times of metabolic stress by utilizing alternative mechanisms of translation initiation. The central hypothesis of this proposal is that translational control of gene expression plays a critical role in early stages of Treg phenotype induction. In order to test this idea, we have done some preliminary experiments using translational profiling, a technique which uses density-based stratification of mRNA into heavy and light fractions based on ribosome content. The heavy fractions contain transcripts undergoing translation most actively. Our preliminary results indicate that rapamycin treatment results in dramatic increase in the amount of Foxp3 mRNA transcripts, but without a significant shift into the heavy fractions. Therefore, our results indicate that increased Foxp3 protein expression seen with rapamycin treatment is driven primarily by increased rate of its transcription. However, we also measured the translational profile of Stat5, one of the known transcription factors essential for Foxp3 expression. Remarkably, our data show that Stat5 transcripts are all shifted into the heavy fractions, while the total amount of Stat5 mRNA isn't significantly increased. Therefore, Stat5 expression appears to fit perfectly our central hypothesis. In the first aim of this proposal we will focus on Stat5 as a case study. We will extend our preliminary results and document the kinetics of Stat5 translation, expression levels of total and phosphorylated Stat5, and its binding to the foxp3 gene. In the second aim we will perform genome-wide translational profiling to identify additional candidate molecules that are involved in driving Treg induction. This mechanism of Treg induction represents a new paradigm in the field, which will compliment other current models that focus primarily on chromatin remodeling in T cell differentiation. Clearly, increased mechanistic understanding of Treg induction is critical to future development of new therapeutic strategies in treatment of a great variety of diseases ranging from autoimmunity to cancer. PUBLIC HEALTH RELEVANCE: CD4 T cells play critical roles in immunity by orchestrating the immune responses against infections and cancer. However, a subset of CD4 T cells, called regulatory T cells, inhibits immune responses. These are critical for preventing autoimmune diseases. Ability to induce antigen-specific regulatory T cells could help to develop new therapeutic strategies for a wide range of diseases. We are starting a novel mechanistic investigation of how regulatory T cells get induced by looking specifically at the role of protein translation control in the cells.